CN111304292A - A genetic screening method beneficial to pig reproduction - Google Patents

Abstract

The invention provides a genetic screening method beneficial to pig reproduction, and relates to the technical field of biomolecules. The excellent screening method for genes beneficial to pig reproduction includes selecting test materials, collecting DNA samples, obtaining primer pairs, PCR amplification, polymorphism detection and artificially synthesizing genes. By extracting the DNA samples of sows, and performing PCR amplification and RFLP analysis, two excellent genes, such as large litter size and strong disease resistance, can be directly obtained, which are beneficial to pig reproduction. The probability of genes is also higher, which greatly reduces the difficulty of gene screening for pig reproduction, and through artificial gene synthesis technology can be obtained. The genes can be inherited in the pig breeding system at the same time, which also makes the selected genes more beneficial to pig breeding.

Description

A genetic screening method beneficial to pig reproduction

technical field

The invention relates to the technical field of biomolecules, in particular to a method for screening good genes for pig reproduction.

Background technique

The pig industry is an important industry in my country's agriculture, and it plays an important role in ensuring the safe supply of meat food. At present, my country's pig industry is transforming from a traditional pig industry to a modern pig industry, whether it is breeding mode, regional layout or production method, Significant changes are taking place in production capacity, but there are still many challenges, such as weak independent innovation ability, prominent food safety problems, increased labor costs, dependence on imported seeds, serious epidemic diseases, environmental protection pressure, and lack of feed resources. However, there are also improvements in independent innovation conditions. , large international market space, steady growth of domestic market, strong government support and other opportunities.

In the pig industry, reproductive traits such as total litter size, piglet uniformity, stillbirth and mummy parity are of great significance to the economic benefits of sow production. Reproductive performance is particularly important in actual production. According to the statistics of some scholars in the early 21st century, it can be found that as the number of litter of sows increases by one, the economic benefit will increase by about 100 yuan on average.

Reproductive traits, as important economic traits in pig production, have low heritability, and it takes a long time to obtain good genes that are beneficial to pig reproduction through conventional breeding methods, and it is difficult to make great genetic progress.

SUMMARY OF THE INVENTION

(1) Technical problems solved

Aiming at the deficiencies of the prior art, the present invention provides a method for screening good genes for pig reproduction, which solves the problem that conventional breeding methods take a long time to screen good genes good for pig reproduction, and it is difficult to obtain good genes.

(2) Technical solutions

In order to achieve the above purpose, the present invention is achieved through the following technical solutions: a genetic screening method beneficial to pig reproduction, which comprises the following steps:

S1 Select test material

First, 800 Sujiang pig sows were selected as the test material;

S2 Collection of DNA samples

DNA samples of ear tissue were extracted from each Sujiang pig sow, divided into two equal parts, and stored at -20℃～-80℃;

S3 get primer pair

The first primer pair was obtained by cluster analysis according to the genes known to have the largest litter size in Meishan pigs, and the second primer pair was obtained by cluster analysis according to the genes known to have strong disease resistance in Landrace pigs;

S4 PCR amplification

Take the two extracted DNA samples as templates, and carry out PCR amplification under the guidance of the first primer pair and the second primer respectively to obtain two PCR amplification products;

S5 polymorphism detection

The polymorphism sequence analysis of the two PCR amplification products was carried out by using RFLP technology, and the results were statistically analyzed to obtain the genes with more litter size and the genes with strong disease resistance in Sujiang pigs;

S6 synthetic gene

Using the principle of gene recombination, the Sujiang pigs have artificially synthesized the genes with more litter size and the genes with strong disease resistance.

Preferably, the PCR reaction system is: 11.5 μl of Taq DNA polymerase, 1.5 μl of 50 ng/μl DNA sample template, 1.0 μl of 10 pmol/L first primer pair or second primer pair, and then supplement the reaction with ultrapure water system to 25 μl.

Preferably, the PCR reaction conditions are: pre-denaturation at 95°C for 4.5 min, then denaturation at 95°C for 30s, annealing at 55.5°C for 30s, extension at 71°C for 30s, 35 cycles, and finally extension at 71°C for 4.5min.

Preferably, the RFLP reaction system is as follows: 8.0 μl of PCR amplification product, 1.0 μl of restriction endonuclease, and 2.0 μl of 10× Buffer dilution solution are added, and then 4.0 μl of ultrapure water is used to supplement the reaction system to 15μl, then heated in a water bath for 3.5h at 35°C to 37°C, and finally detected by 2.0% agarose gel electrophoresis.

Preferably, the 800 Sujiang pig sows are all healthy and disease-free experimental materials.

Preferably, the reproductive shape of the sow mainly includes the number of litter, the number of effective live piglets, the coefficient of primary lesions of piglets, and the like.

(3) Beneficial effects

The present invention provides a genetic screening method beneficial to pig reproduction. Has the following beneficial effects:

1. In the present invention, by extracting the DNA sample of the sow, and performing PCR amplification and RFLP analysis, two kinds of excellent genes, such as large litter size and strong disease resistance, which are beneficial to pig reproduction, can be directly obtained, compared with conventional breeding. With the method, the speed of obtaining good genes is faster, and the probability of obtaining good genes is also higher, which greatly reduces the difficulty of gene screening that is beneficial to pig reproduction.

2. The present invention can synthesize two kinds of excellent genes of large litter size and strong disease resistance through artificial synthetic gene technology, so that the two genes of large litter size and strong disease resistance can be simultaneously in the pig breeding system. Carry out inheritance, so that the offspring of pig breeding can have the characteristics of large litter size and strong disease resistance, and the screened genes are more conducive to the reproduction of pigs.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example:

The embodiment of the present invention provides a genetic screening method beneficial to pig reproduction, which comprises the following steps:

S1 Select test material

First, 800 Sujiang pig sows were selected as the test material, and the number of 800 was selected to increase the number of DNA samples extracted, so as to ensure the accuracy of the test data;

S2 Collection of DNA samples

DNA samples of ear tissue were extracted from each Sujiang pig sow and divided into two equal parts, each of 400 DNA samples. Better preservation of DNA samples, thereby preventing sample damage and failure of genetic screening;

S3 get primer pair

The first primer pair was obtained by cluster analysis based on the genes known to have a large litter size in Meishan pigs. It is known that Meishan pigs have the characteristics of large litter numbers. At the same time, cluster analysis was carried out based on genes known to have strong disease resistance in Landrace pigs. The second primer pair, it is known that Landrace pigs have the characteristics of strong disease resistance;

S4 PCR amplification

Using the two extracted DNA samples as templates, PCR amplification is carried out under the guidance of the first primer pair and the second primer respectively to obtain two PCR amplification products. The gene to be amplified can be amplified by PCR within 2 to 3 hours. the number increased to several million times;

S5 polymorphism detection

The polymorphism sequence analysis of the two PCR amplification products was carried out by using RFLP technology, and the results of statistical analysis were used to obtain the genes with more litter size and the genes with strong disease resistance in Sujiang pigs. The implementation steps of RFLP technology are: The DNA samples are digested with sex endonuclease, and then the DNA fragments are separated by gel electrophoresis, and then the DNA fragments are transferred to the filter membrane, and finally the specific DNA fragments are displayed by the hybridization of radiolabeled probes and the results are analyzed;

S6 synthetic gene

Using the principle of gene recombination, artificially synthesize the gene with more litter size and the gene with strong disease resistance in Sujiang pigs, and artificially synthesize the two genes together, so that the two genes can be inherited in the pig breeding system at the same time, enhancing the The stability of the inheritance of excellent genes that are beneficial to pig reproduction.

The PCR reaction system is: Taq DNA polymerase 11.5μl, 50ng/μl DNA sample template 1.5μl, 10pmol/L first primer pair or second primer pair 1.0μl, and then supplement the reaction system with ultrapure water to 25μl.

The PCR reaction conditions were as follows: pre-denaturation at 95 °C for 4.5 min, followed by denaturation at 95 °C for 30 s, annealing at 55.5 °C for 30 s, extension at 71 °C for 30 s, 35 cycles, and finally extension at 71 °C for 4.5 min.

The RFLP reaction system is: 8.0 μl of PCR amplification product, 1.0 μl of restriction endonuclease, and 2.0 μl of 10× Buffer dilution solution, and then 4.0 μl of ultrapure water to supplement the reaction system to 15 μl, and then water bath The pot was heated in a water bath at 37 °C for 3.5 h, and finally the results of enzyme digestion were detected by 2.0% agarose gel electrophoresis.

The 800 Sujiang pig sows are all healthy and disease-free experimental materials, in order to prevent diseased pigs from affecting the experimental materials, resulting in the failure of genetic screening.

The reproductive shape of sows mainly includes litter size, effective live litter size, and primary disease coefficient of piglets.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, and substitutions can be made in these embodiments without departing from the principle and spirit of the invention and modifications, the scope of the present invention is defined by the appended claims and their equivalents.

Claims (6)

1. A gene excellent screening method beneficial to pig breeding comprises the following steps:

s1 selection of test materials

Firstly, 800 Sujiang sow sows are selected as test materials;

s2 collecting DNA sample

Extracting DNA samples of ear tissues from each Sujiang sow, dividing the DNA samples into two equal parts, and storing the two parts at the temperature of between 20 ℃ below zero and 80 ℃ below zero;

s3 obtaining primer pair

Performing clustering analysis according to the genes with known large litter size of Meishan pigs to obtain a first primer pair, and performing clustering analysis according to the genes with known strong disease resistance of Changbai pigs to obtain a second primer pair;

s4 PCR amplification

Taking the two extracted DNA samples as templates, and respectively carrying out PCR amplification under the guidance of a first primer pair and a second primer pair to obtain two PCR amplification products;

s5 polymorphism detection

Carrying out polymorphic sequence analysis on the two PCR amplification products by using an RFLP technology, and counting the analysis result to obtain a gene with a large litter size of Sujiang pigs and a gene with strong disease resistance;

s6 artificially synthesized gene

By utilizing the principle of gene recombination, the gene with large litter size of Sujiang pigs and the gene with strong disease resistance are artificially synthesized.

2. The method for screening genes favorable for pig breeding according to claim 1, wherein the method comprises the following steps: the PCR reaction system is as follows: 11.5. mu.l of Taq DNA polymerase, 1.5. mu.l of 50 ng/. mu.l of DNA sample template, 1.0. mu.l of 10pmol/L of the first primer pair or the second primer pair, and then supplementing the reaction system to 25. mu.l with ultrapure water.

3. The method for screening genes favorable for pig breeding according to claim 1, wherein the method comprises the following steps: the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 4.5min, denaturation at 95 ℃ for 30s, annealing at 55.5 ℃ for 30s, extension at 71 ℃ for 30s, 35 cycles, and extension at 71 ℃ for 4.5 min.

4. The method for screening genes favorable for pig breeding according to claim 1, wherein the method comprises the following steps: the RFLP reaction system is as follows: mu.l of PCR amplification product is added with 1.0 mu.l of restriction enzyme and 2.0 mu.l of 10 Xbuffer diluent, the reaction system is supplemented with 4.0 mu.l of ultrapure water to 15 mu.l, then the reaction system is heated in water bath for 3.5h at 35-37 ℃ by a water bath kettle, and finally the enzyme digestion result is detected by 2.0% agarose gel electrophoresis.

5. The method for screening genes favorable for pig breeding according to claim 1, wherein the method comprises the following steps: the 800 Sujiang sow sows are all healthy disease-free experimental materials.

6. The method for screening genes favorable for pig breeding according to claim 1, wherein the method comprises the following steps: the breeding shape of the sow mainly comprises the litter size, the effective number born, the piglet birth lesion coefficient and the like.